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1.  Direct Comparison of Au3+ and C60+ Cluster Projectiles in SIMS Molecular Depth Profiling 
The sputtering properties of two representative cluster ion beams in secondary ion mass spectrometry (SIMS), C60+ and Au3+, have been directly compared. Organic thin films consisting of trehalose and dipalmitoylphosphatidylcholine (DPPC) are employed as prototypical targets. The strategy is to make direct comparison of the response of a molecular solid to each type of the bombarding cluster by overlapping the two ion beams onto the same area of the sample surface. The ion beams alternately erode the sample while keeping the same projectile for spectral acquisition. The results from these experiments are important to further optimize the use of cluster projectiles for SIMS molecular depth profiling experiments. For example, Au3+ bombardment is found to induce more chemical damage as well as Au implantation when compared with C60+. Moreover, C60+ is found to be able to remove the damage and the implanted Au effectively. Discussions are also presented on strategies of enhancing sensitivity for imaging applications with cluster SIMS.
doi:10.1016/j.jasms.2006.10.017
PMCID: PMC2000379  PMID: 17118671
2.  Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry 
Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 μm wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 μm and ~40 μm, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.
doi:10.3791/2097
PMCID: PMC3157867  PMID: 20834223
3.  Metal and complementary molecular bioimaging in Alzheimer's disease 
Alzheimer's disease (AD) is the leading cause of dementia in the elderly, affecting over 27 million people worldwide. AD represents a complex neurological disorder which is best understood as the consequence of a number of interconnected genetic and lifestyle variables, which culminate in multiple changes to brain structure and function. These can be observed on a gross anatomical level in brain atrophy, microscopically in extracellular amyloid plaque and neurofibrillary tangle formation, and at a functional level as alterations of metabolic activity. At a molecular level, metal dyshomeostasis is frequently observed in AD due to anomalous binding of metals such as Iron (Fe), Copper (Cu), and Zinc (Zn), or impaired regulation of redox-active metals which can induce the formation of cytotoxic reactive oxygen species and neuronal damage. Metal chelators have been administered therapeutically in transgenic mice models for AD and in clinical human AD studies, with positive outcomes. As a result, neuroimaging of metals in a variety of intact brain cells and tissues is emerging as an important tool for increasing our understanding of the role of metal dysregulation in AD. Several imaging techniques have been used to study the cerebral metallo-architecture in biological specimens to obtain spatially resolved data on chemical elements present in a sample. Hyperspectral techniques, such as particle-induced X-ray emission (PIXE), energy dispersive X-ray spectroscopy (EDS), X-ray fluorescence microscopy (XFM), synchrotron X-ray fluorescence (SXRF), secondary ion mass spectrometry (SIMS), and laser ablation inductively coupled mass spectrometry (LA-ICPMS) can reveal relative intensities and even semi-quantitative concentrations of a large set of elements with differing spatial resolution and detection sensitivities. Other mass spectrometric and spectroscopy imaging techniques such as laser ablation electrospray ionization mass spectrometry (LA ESI-MS), MALDI imaging mass spectrometry (MALDI-IMS), and Fourier transform infrared spectroscopy (FTIR) can be used to correlate changes in elemental distribution with the underlying pathology in AD brain specimens. Taken together, these techniques provide new techniques to probe the pathobiology of AD and pave the way for identifying new therapeutic targets. The current review aims to discuss the advantages and challenges of using these emerging elemental and molecular imaging techniques, and highlight clinical achievements in AD research using bioimaging techniques.
doi:10.3389/fnagi.2014.00138
PMCID: PMC4098123  PMID: 25076902
LA-ICPMS; metals; Alzheimer's disease; bioimaging; MALDI; FTIR
4.  MALDI Imaging Mass Spectrometry of Neuropeptides in Parkinson's Disease 
MALDI imaging mass spectrometry (IMS) is a powerful approach that facilitates the spatial analysis of molecular species in biological tissue samples2 (Fig.1). A 12 μm thin tissue section is covered with a MALDI matrix, which facilitates desorption and ionization of intact peptides and proteins that can be detected with a mass analyzer, typically using a MALDI TOF/TOF mass spectrometer. Generally hundreds of peaks can be assessed in a single rat brain tissue section. In contrast to commonly used imaging techniques, this approach does not require prior knowledge of the molecules of interest and allows for unsupervised and comprehensive analysis of multiple molecular species while maintaining high molecular specificity and sensitivity2. Here we describe a MALDI IMS based approach for elucidating region-specific distribution profiles of neuropeptides in the rat brain of an animal model Parkinson's disease (PD).
PD is a common neurodegenerative disease with a prevalence of 1% for people over 65 of age3,4. The most common symptomatic treatment is based on dopamine replacement using L-DOPA5. However this is accompanied by severe side effects including involuntary abnormal movements, termed L-DOPA-induced dyskinesias (LID)1,3,6. One of the most prominent molecular change in LID is an upregulation of the opioid precursor prodynorphin mRNA7. The dynorphin peptides modulate neurotransmission in brain areas that are essentially involved in movement control7,8. However, to date the exact opioid peptides that originate from processing of the neuropeptide precursor have not been characterized. Therefore, we utilized MALDI IMS in an animal model of experimental Parkinson's disease and L-DOPA induced dyskinesia.
MALDI imaging mass spectrometry proved to be particularly advantageous with respect to neuropeptide characterization, since commonly used antibody based approaches targets known peptide sequences and previously observed post-translational modifications. By contrast MALDI IMS can unravel novel peptide processing products and thus reveal new molecular mechanisms of neuropeptide modulation of neuronal transmission. While the absolute amount of neuropeptides cannot be determined by MALDI IMS, the relative abundance of peptide ions can be delineated from the mass spectra, giving insights about changing levels in health and disease. In the examples presented here, the peak intensities of dynorphin B, alpha-neoendorphin and substance P were found to be significantly increased in the dorsolateral, but not the dorsomedial, striatum of animals with severe dyskinesia involving facial, trunk and orolingual muscles (Fig. 5). Furthermore, MALDI IMS revealed a correlation between dyskinesia severity and levels of des-tyrosine alpha-neoendorphin, representing a previously unknown mechanism of functional inactivation of dynorphins in the striatum as the removal of N-terminal tyrosine reduces the dynorphin's opioid-receptor binding capacity9. This is the first study on neuropeptide characterization in LID using MALDI IMS and the results highlight the potential of the technique for application in all fields of biomedical research.
doi:10.3791/3445
PMCID: PMC3529518  PMID: 22370902
Medicine; Issue 60; Parkinson's disease; L-DOPA induced dyskinesia; striatum; opioid peptides; MALDI Imaging MS
5.  Mass Spectrometry Imaging under Ambient Conditions 
Mass spectrometry reviews  2012;32(3):218-243.
Mass spectrometry imaging (MSI) has emerged as an important tool in the last decade and it is beginning to show potential to provide new information in many fields owing to its unique ability to acquire molecularly specific images and to provide multiplexed information, without the need for labeling or staining. In MSI, the chemical identity of molecules present on a surface is investigated as a function of spatial distribution. In addition to now standard methods involving MSI in vacuum, recently developed ambient ionization techniques allow MSI to be performed under atmospheric pressure on untreated samples outside the mass spectrometer. Here we review recent developments and applications of MSI emphasizing the ambient ionization techniques of desorption electrospray ionization (DESI), laser ablation electrospray ionization (LAESI), probe electrospray ionization (PESI), desorption atmospheric pressure photoionization (DAPPI), femtosecond laser desorption ionization (fs-LDI), laser electrospray mass spectrometry (LEMS), infrared laser ablation metastable-induced chemical ionization (IR-LAMICI), liquid microjunction surface sampling probe mass spectrometry (LMJ-SSP MS), nanospray desorption electrospray ionization (nano-DESI), and plasma sources such as the low temperature plasma (LTP) probe and laser ablation coupled to flowing atmospheric-pressure afterglow (LA-FAPA). Included are discussions of some of the features of ambient MSI including the ability to implement chemical reactions with the goal of providing high abundance ions characteristic of specific compounds of interest and the use of tandem mass spectrometry to either map the distribution of targeted molecules with high specificity or to provide additional MS information in the structural identification of compounds. We also describe the role of bioinformatics in acquiring and interpreting the chemical and spatial information obtained through MSI, especially in biological applications for tissue diagnostic purposes. Finally, we discuss the challenges in ambient MSI and include perspectives on the future of the field.
doi:10.1002/mas.21360
PMCID: PMC3530640  PMID: 22996621
mass spectrometry; imaging; ambient ionization; ionization techniques; review
6.  Molecular sputter depth profiling using carbon cluster beams 
Sputter depth profiling of organic films while maintaining the molecular integrity of the sample has long been deemed impossible because of the accumulation of ion bombardment-induced chemical damage. Only recently, it was found that this problem can be greatly reduced if cluster ion beams are used for sputter erosion. For organic samples, carbon cluster ions appear to be particularly well suited for such a task. Analysis of available data reveals that a projectile appears to be more effective as the number of carbon atoms in the cluster is increased, leaving fullerene ions as the most promising candidates to date. Using a commercially available, highly focused C60q+ cluster ion beam, we demonstrate the versatility of the technique for depth profiling various organic films deposited on a silicon substrate and elucidate the dependence of the results on properties such as projectile ion impact energy and angle, and sample temperature. Moreover, examples are shown where the technique is applied to organic multilayer structures in order to investigate the depth resolution across film-film interfaces. These model experiments allow collection of valuable information on how cluster impact molecular depth profiling works and how to understand and optimize the depth resolution achieved using this technique.
doi:10.1007/s00216-009-2971-x
PMCID: PMC2863088  PMID: 19649771
Molecular depth profiling; Cluster SIMS; Carbon clusters; Cluster ion beams
7.  Disease Biomarkers in Cerebrospinal Fluid of Patients with First-Onset Psychosis 
PLoS Medicine  2006;3(11):e428.
Background
Psychosis is a severe mental condition that is characterized by a loss of contact with reality and is typically associated with hallucinations and delusional beliefs. There are numerous psychiatric conditions that present with psychotic symptoms, most importantly schizophrenia, bipolar affective disorder, and some forms of severe depression referred to as psychotic depression. The pathological mechanisms resulting in psychotic symptoms are not understood, nor is it understood whether the various psychotic illnesses are the result of similar biochemical disturbances. The identification of biological markers (so-called biomarkers) of psychosis is a fundamental step towards a better understanding of the pathogenesis of psychosis and holds the potential for more objective testing methods.
Methods and Findings
Surface-enhanced laser desorption ionization mass spectrometry was employed to profile proteins and peptides in a total of 179 cerebrospinal fluid samples (58 schizophrenia patients, 16 patients with depression, five patients with obsessive-compulsive disorder, ten patients with Alzheimer disease, and 90 controls). Our results show a highly significant differential distribution of samples from healthy volunteers away from drug-naïve patients with first-onset paranoid schizophrenia. The key alterations were the up-regulation of a 40-amino acid VGF-derived peptide, the down-regulation of transthyretin at ~4 kDa, and a peptide cluster at ~6,800–7,300 Da (which is likely to be influenced by the doubly charged ions of the transthyretin protein cluster). These schizophrenia-specific protein/peptide changes were replicated in an independent sample set. Both experiments achieved a specificity of 95% and a sensitivity of 80% or 88% in the initial study and in a subsequent validation study, respectively.
Conclusions
Our results suggest that the application of modern proteomics techniques, particularly mass spectrometric approaches, holds the potential to advance the understanding of the biochemical basis of psychiatric disorders and may in turn allow for the development of diagnostics and improved therapeutics. Further studies are required to validate the clinical effectiveness and disease specificity of the identified biomarkers.
Protein profiles from 179 cerebrospinal fluid samples yield differences between patients with psychotic disorders and healthy volunteers, suggesting that such biomarkers could assist in the early diagnosis of mental illness.
Editors' Summary
Background.
Psychosis is an abnormal mental state characterized by loss of contact with reality, often associated with hallucinations, delusions, personality changes, and disorganized thinking. Psychotic symptoms occur in several psychiatric disorders, including schizophrenia, bipolar disorder, and psychotic depression. It is not clear what the underlying biological abnormalities in the brain are, and whether they are the same for the different psychotic illnesses. The hope is that recent advances in brain imaging and systematic characterization of genetic activity and protein composition in the brain might help to shed light on mental diseases, eventually leading to better diagnosis, treatment, and possibly even prevention.
Why Was This Study Done?
This study was carried out in order to search for biomarkers for psychosis and schizophrenia by comparing the protein composition in the cerebrospinal fluid (the clear body fluid that surrounds the brain and the spinal cord) of patients with different psychotic disorders and normal individuals who served as controls.
What Did the Researchers Do and Find?
The researchers used a technique called surface-enhanced laser desorption ionization mass spectrometry, which allows a comprehensive analysis of the protein composition of a particular sample, on a total of 179 cerebrospinal fluid samples. The samples came from 90 individuals without mental illness who served as controls, 58 people with schizophrenia who were very recently diagnosed and had not yet taken any medication, 16 patients with depression, five patients with obsessive-compulsive disorder, and ten patients with Alzheimer disease. All of the patients gave their informed consent to participate in the study. The researchers found that samples from treatment-naïve schizophrenic patients had a number of characteristic changes compared with samples from control individuals, and that those changes were not found in the patients with other mental illnesses. The researchers then wanted to test whether they would see the same pattern in a separate set of patients with schizophrenia versus controls, which turned out to be the case. Two of the changes in the cerebrospinal fluid that were associated with schizophrenia, namely higher levels of parts of a protein called VGF and lower levels of a protein called transthyretin, were also found in post-mortem brain samples of patients with schizophrenia compared with samples from controls. Lower levels of transthyretin were also found in serum (blood) of first-onset drug naïve schizophrenia patients.
What Do These Findings Mean?
These results suggest that this approach has the potential to find biomarkers for psychosis and, possibly, schizophrenia that might help in the understanding of the molecular basis for these conditions. If shown, in future studies, to be directly involved in causing the disease symptoms, they would be important targets for treatment and prevention efforts, and might also be useful for diagnostic purposes. Overall, there are promising examples, such as this study, suggesting that new molecular techniques can yield fresh insights into psychiatric illnesses such as schizophrenia and other psychotic disorders. Additional studies are needed to confirm the findings presented here and to address many open questions, and would seem well justified given these results.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030428.
MedlinePlus entries on psychosis and schizophrenia
The National Alliance for Research on Schizophrenia and Depression
The National Alliance for the Mentally Ill
The Schizophrenia Society of Canada
Wikipedia entries on psychosis and schizophrenia (note that Wikipedia is an online encyclopedia that anyone can edit)
doi:10.1371/journal.pmed.0030428
PMCID: PMC1630717  PMID: 17090210
8.  Mass spectrometric analysis of glycosphingolipid antigens for NKT cells 
Short Abstract
A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.
Long Abstract
Glycosphingolipids (GSL’s) belong to the glycoconjugate class of biomarcromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1–2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Galα4Galβ4Glcβ1Cer) and isoglobotriaosylceramide (Galα3Galβ4Glcβ1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3–4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7–9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10–15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.
doi:10.3791/4224
PMCID: PMC3664971  PMID: 23628911
Functional glycomics; glycosphingolipids; antigens; natural killer T cells; MALDI-TOF mass spectrometry; LTQ ion trap mass spectrometer
9.  Which is more important in bioimaging SIMS experiments—The sample preparation or the nature of the projectile? 
Applied surface science  2008;255(4):1298-1304.
Sample preparation is central to acquiring meaningful molecule-specific images with SIMS, especially when submicron lateral resolution is involved. The issue is to maintain the distribution of target molecules while attempting to introduce biological cells or tissue into the high vacuum environment of the mass spectrometer. Here we compare freeze-drying, freeze-etching, freeze-fracture and trehalose vitrification as possible strategies for these experiments. The results show that the prospects for successful imaging experiments are greatly improved with all of these methods when using cluster ion bombardment, particularly C60+ ions, not only due to increased sensitivity of this projectiles, but also since it removes contamination overlayers without insult to the underlying chemistry. The emergence of 3-dimensional imaging capabilities also suggests that sample preparation should not perturb the 3-dimensional morphology of the cell, a situation not generally possible during freeze-drying. Hence, sample preparation and projectile type are strongly coupled parameters for bioimaging with mass spectrometry.
doi:10.1016/j.apsusc.2008.05.139
PMCID: PMC2700758  PMID: 19554199
Bioimaging; Cluster SIMS; Freeze-fracture; Freeze-etching; C60; Vitrification
10.  Tissue Imaging Using Nanospray Desorption Electrospray Ionization Mass Spectrometry 
Analytical Chemistry  2011;84(1):141-148.
Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially-resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.
doi:10.1021/ac2021322
PMCID: PMC3259225  PMID: 22098105
imaging mass spectrometry; nanospray desorption electrospray ionization (nano-DESI); rhodamine; tissue; spatial resolution
11.  Simultaneous detection and localization of secondary ions and electrons from single large cluster impacts 
Surface and interface analysis : SIA  2013;45(1):10.1002/sia.4949.
The use of large cluster primary ions (e.g. C60, Au400) in secondary ion mass spectrometry has become prevalent in recent years due to their enhanced emission of secondary ions, in particular, molecular ions (MW ≤ 1500 Da). The co-emission of electrons with SIs was investigated per projectile impact. It has been found that SI and electrons yields increased with increasing projectile energy and size. The use of the emitted electrons from impacts of C60 for localization has been demonstrated for cholesterol deposited on a copper grid. The instrumentation, methodologies, and results from these experiments are presented.
doi:10.1002/sia.4949
PMCID: PMC3807816  PMID: 24163488
Electron Emission Microscope; Cluster SIMS; Electron Emission; Localization
12.  Targeted Multiplex Imaging Mass Spectrometry in Transmission Geometry for Subcellular Spatial Resolution 
Targeted multiplex Imaging Mass Spectrometry utilizes several different antigen-specific primary antibodies, each directly labeled with a unique photocleavable mass tag, to detect multiple antigens in a single tissue section. Each photocleavable mass tag bound to an antibody has a unique molecular weight and can be readily ionized by laser desorption ionization mass spectrometry. This manuscript describes a mass spectrometry method that allows imaging of targeted single cells within tissue using transmission geometry laser desorption ionization mass spectrometry. Transmission geometry focuses the laser beam on the back side of the tissue placed on a glass slide, providing a 2 μm diameter laser spot irradiating the biological specimen. This matrix-free method enables simultaneous localization at the sub-cellular level of multiple antigens using specific tagged antibodies. We have used this technology to visualize the co-expression of synaptophysin and two major hormones peptides, insulin and somatostatin, in duplex assays in beta and delta cells contained in a human pancreatic islet.
doi:10.1007/s13361-012-0563-z
PMCID: PMC3624063  PMID: 23397138
Targeted multiplex mass spectrometry imaging; laser desorption ionization mass spectrometry; transmission geometry; photocleavable mass tag; antibodies; immunohistochemistry; sub-cellular spatial resolution
13.  Lipid Imaging with Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) 
Biochimica et biophysica acta  2011;1811(11):976-990.
Fundamental advances in secondary ion mass spectrometry (SIMS) now allow for the examination and characterization of lipids directly from biological materials. The successful application of SIMS-based imaging in the investigation of lipids directly from tissue and cells are demonstrated. Common complications and technical pitfalls are discussed. In this review, we examine the use of cluster ion sources and cryogenically compatible sample handling for improved ion yields and to expand the application potential of SIMS. Methodological improvements, including pre-treating the sample to improve ion yields and protocol development for 3-dimensional analyses (i.e. molecular depth profiling), are also included in this discussion. New high performance SIMS instruments showcasing the most advanced instrumental developments, including tandem MS capabilities and continuous ion beam compatibility, are described and the future direction for SIMS in lipid imaging is evaluated.
doi:10.1016/j.bbalip.2011.05.007
PMCID: PMC3199347  PMID: 21664291
ToF-SIMS; lipids; cluster sources; sample preparation; C60+QSTAR; J105 3D Chemical Imager and imaging mass spectrometry (IMS)
14.  Molecular Depth Profiling using a C60 Cluster Beam: the Role of Impact Energy 
Molecular depth profiling of organic overlayers was performed using a mass selected C60 ion beam in conjunction with time-of-flight (TOF-SIMS) mass spectrometry. The characteristics of sputter depth profiles acquired for a 300-nm Trehalose film on silicon were studied as a function of the kinetic impact energy of the projectile ions. The results are interpreted in terms of a simple model describing the balance between sputter erosion and ion induced chemical damage. It is shown that the efficiency of the projectile to clean up the fragmentation debris produced by its own impact represents a key parameter governing the success of molecular depth profile analysis.
doi:10.1021/jp8049763
PMCID: PMC2662745  PMID: 19855815
15.  Mass spectrometry imaging with high resolution in mass and space 
Histochemistry and Cell Biology  2013;139(6):759-783.
Mass spectrometry (MS) imaging links molecular information and the spatial distribution of analytes within a sample. In contrast to most histochemical techniques, mass spectrometry imaging can differentiate molecular modifications and does not require labeling of targeted compounds. We have recently introduced the first mass spectrometry imaging method that provides highly specific molecular information (high resolution and accuracy in mass) at cellular dimensions (high resolution in space). This method is based on a matrix-assisted laser desorption/ionization (MALDI) imaging source working at atmospheric pressure which is coupled to an orbital trapping mass spectrometer. Here, we present a number of application examples and demonstrate the benefit of ‘mass spectrometry imaging with high resolution in mass and space.’ Phospholipids, peptides and drug compounds were imaged in a number of tissue samples at a spatial resolution of 5–10 μm. Proteins were analyzed after on-tissue tryptic digestion at 50-μm resolution. Additional applications include the analysis of single cells and of human lung carcinoma tissue as well as the first MALDI imaging measurement of tissue at 3 μm pixel size. MS image analysis for all these experiments showed excellent correlation with histological staining evaluation. The high mass resolution (R = 30,000) and mass accuracy (typically 1 ppm) proved to be essential for specific image generation and reliable identification of analytes in tissue samples. The ability to combine the required high-quality mass analysis with spatial resolution in the range of single cells is a unique feature of our method. With that, it has the potential to supplement classical histochemical protocols and to provide new insights about molecular processes on the cellular level.
doi:10.1007/s00418-013-1097-6
PMCID: PMC3656243  PMID: 23652571
Mass spectrometry imaging; Tissue analysis; High-resolution mass spectrometry; Accurate mass measurements; Mass spectrometry-based histology; MALDI mass spectrometry
16.  Surface characterization of biological nanodomains using NP-ToF-SIMS 
Surface and interface analysis : SIA  2013;45(1):10.1002/sia.4901.
This paper describes the application of nanoparticle bombardment with time-of-flight secondary ion mass spectrometry (NP-ToF-SIMS) for the analysis of native biological surfaces for the case of sagittal sections of mammalian brain tissue. The use of high energy, single nanoparticle impacts (e.g. 520 keV Au400) permits desorption of intact lipid molecular ions, with enhanced molecular ion yield and reduced fragmentation. When coupled with complementary molecular ion fragmentation and exact mass measurement analysis, high energy nanoparticle probes (e.g. 520 keV Au400 NP) provide a powerful tool for the analysis of the lipid components from native brain sections without the need for surface preparation and with ultimate spatial resolution limited to the desorption volume per impact (~103 nm3).
doi:10.1002/sia.4901
PMCID: PMC3808454  PMID: 24163489
NP-ToF-SIMS; MALDI-FT-ICR-MS
17.  Investigation of Different Hierarchal Clustering Approaches for Protein Identification Directly from Tissue Section in a MALDI Imaging Experiment 
Mass spectrometry imaging (MSI) allows for the correlation of spatial localization and chemical information directly from biological surfaces. A data set can contain thousands of ion signals with varying degrees of co-localisation. Ion mobility separation based on travelling wave technology can be utilized to add specificity to the MSI experiment. This leads to highly complex data sets that necessitate the need for advanced automated computerized processing. Here, we investigate the use of different Hierarchal Clustering Analysis (HCA) methods to aid the analysis of digested tissue sections by clustering ion images based on correlation. Four proteins digests from BSA, Phosphorylase B, ADH and Enolase were spotted forming a 6x6 array comprising four 4x4 overlapping squares. Mouse fibrosarcoma model tissue sections were washed and on-tissue tryptic digested overnight. Matrix was applied evenly in several coats. Data were acquired using MALDI SYNAPT G1 and G2-S instruments in MS mode with tri-wave ion guide optics to separate ions according to their ionic mobility. The acquisition mass range was from 700–3,000 Da. Data were processed and visualized using High Definition Imaging MALDI software. Data reduction is initially achieved by peak picking using multidimensional (m/z and drift time) detection algorithms. A second step aims at generating ion distributions images comprising x,y coordinates and a third step correlates all processed ion distributions using Pearson product-moment algorithms. Different HCA methods were assessed in terms of their ability to cluster peaks from the tryptic digest imaging data set into related peptide groups, which can be used for PMF protein identification. The methods were also evaluated in terms of the time required to complete the analysis and number of hierarchal levels created. Top-down K-medoid HCA was applied to the fibrosarcoma tissue data. It successfully clustered tryptic peptides with multiple protein identifications from a complex digested tissue section.
PMCID: PMC4162203
18.  A database application for pre-processing, storage and comparison of mass spectra derived from patients and controls 
BMC Bioinformatics  2006;7:403.
Background
Statistical comparison of peptide profiles in biomarker discovery requires fast, user-friendly software for high throughput data analysis. Important features are flexibility in changing input variables and statistical analysis of peptides that are differentially expressed between patient and control groups. In addition, integration the mass spectrometry data with the results of other experiments, such as microarray analysis, and information from other databases requires a central storage of the profile matrix, where protein id's can be added to peptide masses of interest.
Results
A new database application is presented, to detect and identify significantly differentially expressed peptides in peptide profiles obtained from body fluids of patient and control groups. The presented modular software is capable of central storage of mass spectra and results in fast analysis. The software architecture consists of 4 pillars, 1) a Graphical User Interface written in Java, 2) a MySQL database, which contains all metadata, such as experiment numbers and sample codes, 3) a FTP (File Transport Protocol) server to store all raw mass spectrometry files and processed data, and 4) the software package R, which is used for modular statistical calculations, such as the Wilcoxon-Mann-Whitney rank sum test. Statistic analysis by the Wilcoxon-Mann-Whitney test in R demonstrates that peptide-profiles of two patient groups 1) breast cancer patients with leptomeningeal metastases and 2) prostate cancer patients in end stage disease can be distinguished from those of control groups.
Conclusion
The database application is capable to distinguish patient Matrix Assisted Laser Desorption Ionization (MALDI-TOF) peptide profiles from control groups using large size datasets. The modular architecture of the application makes it possible to adapt the application to handle also large sized data from MS/MS- and Fourier Transform Ion Cyclotron Resonance (FT-ICR) mass spectrometry experiments. It is expected that the higher resolution and mass accuracy of the FT-ICR mass spectrometry prevents the clustering of peaks of different peptides and allows the identification of differentially expressed proteins from the peptide profiles.
doi:10.1186/1471-2105-7-403
PMCID: PMC1594579  PMID: 16953879
19.  Matrix-Assisted Laser Desorption Ionization Imaging Mass Spectrometry: In Situ Molecular Mapping 
Biochemistry  2013;52(22):10.1021/bi301519p.
Matrix-assisted laser desorption ionization imaging mass spectrometry (IMS) is a relatively new imaging modality that allows mapping of a wide range of biomolecules within a thin tissue section. The technology uses a laser beam to directly desorb and ionize molecules from discrete locations on the tissue that are subsequently recorded in a mass spectrometer. IMS is distinguished by the ability to directly measure molecules in situ ranging from small metabolites to proteins, reporting hundreds to thousands of expression patterns from a single imaging experiment. This article reviews recent advances in IMS technology, applications, and experimental strategies that allow it to significantly aid in the discovery and understanding of molecular processes in biological and clinical samples.
doi:10.1021/bi301519p
PMCID: PMC3864574  PMID: 23259809
20.  Label free biochemical 2D and 3D imaging using secondary ion mass spectrometry 
Time-of-flight Secondary ion mass spectrometry (ToF-SIMS) provides a method for the detection of native and exogenous compounds in biological samples on a cellular scale. Through the development of novel ion beams the amount of molecular signal available from the sample surface has been increased. Through the introduction of polyatomic ion beams, particularly C60, ToF-SIMS can now be used to monitor molecular signals as a function of depth as the sample is eroded thus proving the ability to generate 3D molecular images. Here we describe how this new capability has led to the development of novel instrumentation for 3D molecular imaging while also highlighting the importance of sample preparation and discuss the challenges that still need to be overcome to maximise the impact of the technique.
doi:10.1016/j.cbpa.2011.05.016
PMCID: PMC3181283  PMID: 21664172
21.  Molecular Depth Profiling by Wedged Crater Beveling 
Analytical chemistry  2011;83(16):6410-6417.
Time-of-flight secondary ion mass spectrometry and atomic force microscopy are employed to characterize a wedge-shaped crater eroded by a 40keV C60+ cluster ion beam on an organic film of Irganox 1010 doped with Irganox 3114 delta layers. From an examination of the resulting surface, the information about depth resolution, topography and erosion rate can be obtained as a function of crater depth for every depth in a single experiment. It is shown that when measurements are performed at liquid nitrogen temperature, a constant erosion rate and reduced bombardment induced surface roughness is observed. At room temperature, however, the erosion rate drops by ~1/3 during the removal of the 400 nm Irganox film and the roughness gradually increased to from 1 nm ~4 nm. From SIMS lateral images of the beveled crater and AFM topography results, depth resolution was further improved by employing glancing angles of incidence and lower primary ion beam energy. Sub-10 nm depth resolution was observed under the optimized conditions on a routine basis. In general, we show that the wedge-crater beveling is an important tool for elucidating the factors that are important for molecular depth profiling experiments.
doi:10.1021/ac201502w
PMCID: PMC3158663  PMID: 21744861
22.  Rapid Profiling of Bovine and Human Milk Gangliosides by Matrix-Assisted Laser Desorption/Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry 
Gangliosides are anionic glycosphingolipids widely distributed in vertebrate tissues and fluids. Their structural and quantitative expression patterns depend on phylogeny and are distinct down to the species level. In milk, gangliosides are exclusively associated with the milk fat globule membrane. They may participate in diverse biological processes but more specifically to host-pathogen interactions. However, due to the molecular complexities, the analysis needs extensive sample preparation, chromatographic separation, and even chemical reaction, which makes the process very complex and time-consuming. Here, we describe a rapid profiling method for bovine and human milk gangliosides employing matrix-assisted desorption/ionization (MALDI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry (MS). Prior to the analyses of biological samples, milk ganglioside standards GM3 and GD3 fractions were first analyzed in order to validate this method. High mass accuracy and high resolution obtained from MALDI FTICR MS allow for the confident assignment of chain length and degree of unsaturation of the ceramide. For the structural elucidation, tandem mass spectrometry (MS/MS), specifically as collision-induced dissociation (CID) and infrared multiphoton dissociation (IRMPD) were employed. Complex ganglioside mixtures from bovine and human milk were further analyzed with this method. The samples were prepared by two consecutive chloroform/methanol extraction and solid phase extraction. We observed a number of differences between bovine milk and human milk. The common gangliosides in bovine and human milk are NeuAc-NeuAc-Hex-Hex-Cer (GD3) and NeuAc-Hex-Hex-Cer (GM3); whereas, the ion intensities of ganglioside species are different between two milk samples. Kendrick mass defect plot yields grouping of ganglioside peaks according to their structural similarities. Gangliosides were further probed by tandem MS to confirm the compositional and structural assignments. We found that only in human milk gangliosides was the ceramide carbon always even numbered, which is consistent with the notion that differences in the oligosaccharide and the ceramide moieties confer to their physiological distinctions.
doi:10.1016/j.ijms.2010.10.020
PMCID: PMC3158620  PMID: 21860602
Ganglioside; Bovine milk; Human milk; Matrix-assisted laser desorption/ionization; Fourier transform ion cyclotron resonance; Kendrick mass defect
23.  Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions 
Comparative analysis of Erk phosphorylation suggests a mixed strategy for measuring phospho-form distributions. Four methods were compared for quantifying the proportion of Erk2 ‘phospho-forms' in differentially phosphorylated samples, revealing excellent agreement between mass spectrometry and nuclear magnetic resonance, but significant discrepancies with western blots.
We compared four methods for quantifying the proportions of phospho-forms, or ‘phospho-form distribution', of a multiply phosphorylated protein, using as an example the MAP kinase Erk2, with two principal phosphorylation sites.Measurements by mass spectrometry (MS) and by nuclear magnetic resonance (NMR) agreed to within 10%, but phospho-specific antibody measurements exhibited semi-quantitative discrepancies with these, sometimes suggesting reverse trends to those found by the biophysical methods.NMR revealed under our conditions that Erk was phosphorylated on four, not two, sites.A combination of peptide-based MS and protein-based MS provided an optimum strategy for determining the 16=24 member phospho-form distribution.
Protein post-translational modification is one of the most significant regulatory mechanisms in cellular physiology, and protein phosphorylation is the most widely studied of these. An individual molecule may be phosphorylatable on multiple residues, allowing it to exist in a multiplicity of combinatorial patterns of modification; with n-sites, there may be 2n such ‘phospho-forms'. Recent work on many distinct types of proteins—ion channels, signalling enzymes, transcription factors, co-activators, circadian clock components—has shown that different phospho-forms may have different downstream effects. The impact of multisite phosphorylation, therefore, is determined by the proportions of the various phospho-forms that are present in the molecular population of the given protein. This ‘phospho-form distribution' is dynamically and collectively regulated by the opposing actions of the relevant kinases and phosphatases. This presents a more challenging perspective than is depicted in the typical cartoon diagram, in which is shown only a single phospho-form, usually the maximally phosphorylated one, and the underlying dynamics of modification and demodification is left implicit.
The present paper sets out to bring this perspective of phospho-forms and phospho-form distributions to a wider biological audience, to compare current methods for measuring them and to discuss the challenges in developing a general strategy applicable to the kinds of proteins typically found in cellular physiology. We chose to analyse the 42 kDa mitogen-activated protein (MAP) kinase Erk2 (Erk). Erk is a paradigmatic signalling protein that is phosphorylated on T and Y residues in a TEY motif within its kinase-activation loop. Because these sites are so close together, multiple methods may be used to detect phospho-forms and we compared four: western blots with phospho-specific antibodies; peptide-based liquid chromatography (LC)/mass spectrometry (MS), in which proteins are first digested into peptide (pepMS); protein-based LC/MS with intact proteins (proMS); and nuclear magnetic resonance spectroscopy (NMR). To provide a stringent comparison, we used specific kinases and phosphatases to prepare four samples of Erk in distinct states of phosphorylation and sought to determine the proportion of each of the four phospho-forms in each of the four samples.
We found excellent agreement, to within 10%, between the various biophysical methods, pepMS, proMS and NMR, despite the experiments being carried out in three different laboratories on two different continents. NMR also revealed the presence of two additional phosphorylations on one of the samples, which we identified by MS as serine phosphorylations. We determined most of the corresponding 16 member phospho-form distribution using a combination of pepMS and proMS. To our surprise, however, we found significant semi-quantitative discrepancies between the biophysical and the immunological methods, despite using the LICOR method of ratiometric fluorescent imaging for western blotting. For instance, a phospho-specific antibody may indicate that sample one has a higher proportion of a certain phospho-form than sample two, but pepMS measurements may sometimes indicate the reverse (compare Figures 1D and 2C). We found similar discrepancies with an alternative set of samples, prepared differently (compare Figure 3A and B), and after spiking western blots with whole-cell lysate (Figure 3C) and after using chemiluminescence and CCD imaging in place of fluorescent detection.
Antibodies are usually characterised for the purposes of quantitative measurement by titration against the same sample, but molecular recognition between antibody and antigen is an emergent property of the biological context. In the comparisons made here, the same phospho-form is being examined in different samples and hence in the context of different phospho-form distributions. The antibody sees not only the phospho-epitope that is its nominal target but also differing amounts of other phospho-epitopes against which it may have a range of cross-reactivities. Such a context-dependent interaction may be one reason for the surprising discrepancies that were found. If so, it exemplifies one of our central themes: it is not any single phospho-form that determines the downstream response, in this case of an antibody; it is the entire phospho-form distribution. While antibodies remain unrivalled for protein detection sensitivity amidst complex cellular backgrounds, our results suggest that care is required in using them for quantitative comparisons of post-translational modifications.
If sites can be localised to a single peptide, pepMS with both LC and MS offers good opportunities for separating distinct phospho-forms, including isobaric ones having the same molecular weight. However, such measurements are not quantitative because distinct phospho-forms may ionise and ‘fly' with different efficiencies; isotopically-labelled phospho-peptide internal standards are required for accurate measurements. Moreover, sites on distinct peptides can no longer be correlated with each other. Measurements with intact protein, as in proMS, avoid both problems but are also less sensitive to LC separation, allowing only the distribution of isobaric forms to be determined. The use of multiple samples, prepared with specific phosphatases, can also be informative. The combination of pepMS and proMS offers a hybrid strategy that represents a good balance between coverage and resolution and holds out promise as a general approach for proteins with small numbers of sites. NMR has certain inherent limitations, being unable to detect correlations between phosphorylations that are not close together and being better able to detect phosphorylation on serine and threonine than on tyrosine. However, it has the advantage, as does proMS, of not being biased by prior expectations as to where modifications are expected, illustrated by its uncovering of the two additional phosphorylations on Erk. While NMR's specialised requirements make it less feasible as a general methodology, it holds out the promise of real-time measurements both in vitro and in intact cells.
The problem of quantifying phospho-form distribution remains very challenging as the number of phosphorylated sites increases but quantitative information is now becoming available for proteins such as Erk that are commonly encountered in cellular physiology.
The functional impact of multisite protein phosphorylation can depend on both the numbers and the positions of phosphorylated sites—the global pattern of phosphorylation or ‘phospho-form'—giving biological systems profound capabilities for dynamic information processing. A central problem in quantitative systems biology, therefore, is to measure the ‘phospho-form distribution': the relative amount of each of the 2n phospho-forms of a protein with n-phosphorylation sites. We compared four potential methods—western blots with phospho-specific antibodies, peptide-based liquid chromatography (LC) and mass spectrometry (MS; pepMS), protein-based LC/MS (proMS) and nuclear magnetic resonance spectroscopy (NMR)—on differentially phosphorylated samples of the well-studied mitogen-activated protein kinase Erk2, with two phosphorylation sites. The MS methods were quantitatively consistent with each other and with NMR to within 10%, but western blots, while highly sensitive, showed significant discrepancies with MS. NMR also uncovered two additional phosphorylations, for which a combination of pepMS and proMS yielded an estimate of the 16-member phospho-form distribution. This combined MS strategy provides an optimal mixture of accuracy and coverage for quantifying distributions, but positional isomers remain a challenging problem.
doi:10.1038/msb.2011.15
PMCID: PMC3097084  PMID: 21487401
mass spectrometry, multisite phosphorylation; NMR; phospho-form distribution; phospho-specific antibodies
24.  Isotopologue Distributions of Peptide Product Ions by Tandem Mass Spectrometry: Quantitation of Low Levels of Deuterium Incorporation1 
Analytical biochemistry  2007;367(1):40-48.
Protonated molecular peptide ions and their product ions generated by tandem mass spectrometry appear as isotopologue clusters due to the natural isotopic variations of carbon, hydrogen, nitrogen, oxygen and sulfur. Quantitation of the isotopic composition of peptides can be employed in experiments involving isotope effects, isotope exchange, isotopic labeling by chemical reactions, and studies of metabolism by stable isotope incorporation. Both ion trap and quadrupole-time of flight mass spectrometry are shown to be capable of determining the isotopic composition of peptide product ions obtained by tandem mass spectrometry with both precision and accuracy. Tandem mass spectra obtained in profile-mode of clusters of isotopologue ions are fit by non-linear least squares to a series of Gaussian peaks (described in the accompanying manuscript) which quantify the Mn/M0 values which define the isotopologue distribution (ID). To determine the isotopic composition of product ions from their ID, a new algorithm that predicts the Mn/M0 ratios is developed which obviates the need to determine the intensity of all of the ions of an ID. Consequently a precise and accurate determination of the isotopic composition a product ion may be obtained from only the initial values of the ID, however the entire isotopologue cluster must be isolated prior to fragmentation. Following optimization of the molecular ion isolation width, fragmentation energy and detector sensitivity, the presence of isotopic excess (2H, 13C, 15N, 18O) is readily determined within 1%. The ability to determine the isotopic composition of sequential product ions permits the isotopic composition of individual amino acid residues in the precursor ion to be determined.
doi:10.1016/j.ab.2007.03.036
PMCID: PMC2153461  PMID: 17559791
isotopologue distribution; mass isotopomer distribution; tandem mass spectrometry; deuterium incorporation; isotopic excess; isotope quantitation; H/D exchange; protein turnover
25.  Detection of Carbohydrates and Steroids by Cation-Enhanced Nanostructure-Initiator Mass Spectrometry (NIMS) for Biofluid Analysis and Tissue Imaging 
Analytical chemistry  2010;82(1):121-128.
Nanostructure-initiator mass spectrometry (NIMS) is a highly sensitive, matrix-free technique that is well suited for biofluid analysis and imaging of biological tissues. Here we provide a new technical variation of NIMS to analyze carbohydrates and steroids, molecules that are challenging to detect with traditional mass spectrometric approaches. Analysis of carbohydrates and steroids was accomplished by spray depositing NaCl or AgNO3 on the NIMS porous silicon surface to provide a uniform environment rich with cationization agents prior to desporption of the fluorinated polymer initiator. Laser desorption/ionization of the ion-coated NIMS surface allowed for Na+ cationization of carbohydrates and Ag+ cationization of steroids. The reliability of the approach is quantitatively demonstrated with a calibration curve over the physiological range of glucose and cholesterol concentrations in human serum (1 – 200 μM). Additionally we illustrate the sensitivity of the method by showing its ability to detect carbohydrates and steroids down to the 800-amol and 100-fmol levels, respectively. The technique developed is well suited for tissue imaging of biologically significant metabolites such as sucrose and cholesterol. To highlight its applicability, we used cation-enhanced NIMS to image the distribution of sucrose in a Gerbera jamesonii flower stem and the distribution of cholesterol in a mouse brain. The flower stem and brain sections were placed directly on the ion-coated NIMS surface without further preparation and analyzed directly. The overall results reported underscore the potential of NIMS to analyze and image chemically diverse compounds that have been traditionally challenging to observe with mass spectrometry-based techniques.
doi:10.1021/ac9014353
PMCID: PMC2802282  PMID: 19961200

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